I. Field of the Invention
The invention relates generally to the field of plant monolignol synthesis, monolignol transport, and lignin polymerization genes and polypeptides encoded by such genes, and the use of such polynucleotide and polypeptide sequences for controlling plant phenotype. The invention specifically provides polynucleotide and polypeptide sequences isolated from Eucalyptus and Pinus species and sequences related thereto.
II. Description of the Related Art
Lignin, a highly hydrophobic and crosslinked phenolic polymer, is a major component of the xylem of plants, especially woody plants such as angiosperm and gymnosperm trees. Lignin is composed of phenylpropanoid units derived from three cinnamyl alcohols, p-coumaroyl, coniferyl and sinapyl alcohol (each differing in the degree of methoxylation at the C3 and C5 positions of the aromic ring), although other phenolics can be incorporated. See, e.g., Sederoff et al. (1999) Curr. Opin. Plant Biol. 2:145-52. These alcohols are also known as monolignols.
Lignin is deposited within the cellulose framework of plant cell secondary cell walls by intussusception. The amount of ligninification varies among plant groups and species, cells and even between different parts of the same plant cell. See, e.g., T. T. Kozlowski and S. G. Pallardy (eds.), 1997, Physiology of Woody Plants, Academic Press, San Diego, Calif. For example, gymnosperm tracheids and angiosperm vessels are heavily lignified; whereas fiber tracheids and libriform fibers of angiosperms show little deposition of lignin. Besides its role as a structural component, lignin facilitates water transport, impedes the degradation of cell wall polysaccharides, and protects against disease-causing organisms, insects and other herbivores.
In contrast, lignin inhibits commercial exploitation of plants by impacting the structural properties of wood and wood products, as well as, the nutritional quality and digestibility of plants. Heavily lignified wood can significantly increase the cost of preparing fiber and wood products. For example, in order to make may grades of paper, it is necessary to remove much of the lignin content from the fiber network of wood. The removal of lignin during the pulping process is expensive, consumes large quantities of chemicals and energy, and potentially environmentally hazardous. Moreover, the difficulty of lignin extraction is relative to the complexity and heterogeneity of the polymer—lignin from gymnosperms, consisting mainly of guaiacyl subunits, is realatively more difficult to extract using Kraft delignification than lignin from angiosperms, consisting of both guaiacyl and syringyl subunits.
Likewise, heavily lignified plants are of poor nutritional quality as such plants generally have low levels of digestibility. As lignin is intimately associated with the cell wall polysaccharides of forages, it interferes with the digestion of those carbohydrates and hemicellulose by limiting their availability to enzymes.
The modulation of lignin content in plants by genetic engineering is an extremely powerful technique by which to ameliorate these negative plant qualities. See, e.g., Dean et al. (1997) Adv. Biochem. Eng. Biotechnol. 57:1-44. The control of lignin synthesis has applications such as the alteration of wood properties and, in particular, lumber and wood pulp properties. For example, wood pulp quality can be effected by increasing or decreasing the quantity or quality of lignin, cellulose, and nonlignin cell wall phenolics. Modulating lignin synthesis in a plant can also engineer functionally tailored lumber having increased or decreased dimensional stability, tensile strength, shear strength, compression strength, stiffness, hardness, spirility, shrinkage, weight, density and specific gravity.
A. The Lignification Process
The lignification process encompasses the biosynthesis of monolignols, their transport to the cell wall, and their polymerization into lignin. The lignification process has been extensively researched. However, new investigations have prompted commentators to note “that the traditional scheme of the lignin pathway is wrong in some respects.” Baucher et al., Crit. Rev. Biochem. Mol. Biol. 38(4):305-50 (2003). Importantly, skilled artisans recognize both their “limited understanding of the chemistry and biochemistry of the plant cell walls” and their ability to predict how specific genetic modifications will influence the ligninfication process. Baucher et al., supra.
Monolignol synthesis, i.e., that of p-coumaroyl, coniferyl and sinapyl alcohols, occurs through a complex series of reactions beginning with the amino acid phenylalanine and catalyzed by a number of multifunctional enzymes. See, e.g., Boerjan et al. (2003) Ann. Rev. Plant Biol. 54:519-46. In brief, the lignification process begins with the deamination of phenylalanine to form cinnamic acid. Cinnamic acid is modified by hydroxylation of the ring, subsequent methoxylation, and reduction of the modified cinnamic acids to cinnamyl alcohols, the monolignol precursors for lignin.
Specifically, the proteins cinnamyl alcohol dehydrogenase (CAD), caffeoyl-CoA O-methyl-transferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamic acid 4-hydroxylase (C4H), p-coumarate 3-hydroxylase (C3H), 4-coumarate:CoA ligase (4CL), caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), ferulate 5-hydroxylase (F5H), hydroxycinnamoyl-CoA:shikimate/quinate (HCT), laccase (LAC), phenylalanine ammonia-lyase (PAL), peroxidase (POX), and sinapyl alcohol dehydrogenase (SAD), each catalyze a reaction in the lignification process. See Humphreys J. M. and C. Chapple, Curr. Opin. Plant Biol. 5(3):224-229 (2002).
In addition to enzymes responsible for monolignol and lignin synthesis, specific enzymes assist in the export of lignin precursors from the cell cytoplasm to the secondary cell wall. Monolignols and monolignol precursors are subsequently chemically modified to assist in transportation and storage, by an array of cellular enzymes. See, e.g., Hosokawa et al., Plant Cell Physiol. 42(9):959-68 (2001). For example, the enzymes coniferol glucosyl transferase (CGT) and beta-glucosidase (CBG) may assist in exportation of coniferyl alcohol and coniferin.
Likewise, following the exportation process, enzymes are needed to catalyze the final steps of the polymerization process. The formation of the lignin macromolecule results from the oxidative coupling between a monolignol and the growing lignin polymer. See, e.g., R. Hatfield and W. Vermerris (2001) Plant Physiol. 126:1351-57. The enzymes phenolase (PNL), peroxidase (POX), laccase (LAC also known as multicopper oxidase) and other phenol oxydases catalyze the polymerization of monolignils into lignin.
Despite the degree of knowledge of the lignification process, significant uncertainties remain. For example, little is known of the storage of monolignols before they are transported to the cell wall. Similarly, skilled artisans generally do not know the subcellular localizations of most enzymes which catalyze lignification. Moreover, as discussed above, revisions of the lignification process have recently occurred as the result of new invesitigations into enzyme substate specificity and kinetics. Accordingly, new techniques are needed to investigate the role of specific enzymes in the lignification process.
B. Genetic Engineering of Plant Lignin Content
Previously, researchers have attempted to modulate the lignin content through the construction of transgenic plants. See, e.g., Baucher et al., supra. Although this research confirmed some of the recent revisions of the lignification process, it “has also opened up new research areas” and “posed new questions” for lignin research.
1. Up- and Down-Regulation of PAL
PAL catalyzes the non-oxidative deamination of phenylalanine to cinnamic acid. As this is the first step of the phenylpropanoid pathway, the reduction of PAL activity leads to a wide variety of abnormal phenotypes. See Elkind et al., Proc. Natl. Acad. Sci. U.S.A. 87:9057-9061 (1990). Transgenic tobacco plants were stunted, had abnormal leaves, reduced lignin content, and high incidence of fungal infection than wild-type plants. See Elkind et al., supra; Maher et al., Proc. Natl. Acad. Sci. U.S.A. 91:7802-7806 (1994).
2. Up- and Down-Regulation of C4H
Hydroxylation of cinnamic acid is catylzyed by C4H, a cytochrome P450-linked monooxygenase. In transgenic tobacco plants, overexpression of C4H had no effect on lignin content nor composition. See Sewalt et al., Plant Physiol. 115:41-50 (1997). In contrast, down-regulation of C4H led to decrease lignin content and altered lignin composition. See Sewalt et al., supra.
3. Down-Regulation of OMT
Transgenic plants which down-regulate OMT have been produced. See Zhong et al., Plant Physiol. 124:536-577 (2000). These plants demonstrated decreased lignin content primarily due to the reduction of both guaiacyl and syringyl subunits. Additionally, although the transgenic poplar were not affected in either growth or morphology, its lignin was much less cross-linked that wild-type poplar.
4. Up- and Down-Regulation of F5H
F5H was renamed coniferaldehyde 5-hydroxylase (Cald5H) upon the realization that it preferentially catalyzes the 5-hydroxylation of the cinnammaldehydes. See Baucher et al., supra. An Arabidopis mutant deficient in F5H reportedly produced lignin deficient in syringyl subunits. See Chapple et al., Plant Cell 4:1413-1424 (1992). Likewise, overexpression of a heterologous isoform of F5H in aspen produced an altered lignin compostition but failed to impact lignin content.
5. Down-Regulation of 4CL
Transgenic plants with reduced 4CL activity have been produced in aspen. See Hu et al., Nature Biotechnol. 17:808-812 (1999). Although these transgenic plants had decreased lignin content, normal cellular morphology and an increased growth rate, the apsens did not show any difference in the lignin composition. Additionally, the transgenic aspens possessed an increase in the amount of nonlignin cell wall phenolics.
6. Down-Regulation of CCR
CCR catalyzes a potential control point in the lignification process, namely the reduction of hydroxycinnamoyl-CoA thioesters to the corresponding aldehydes. Trangenic tobacco plants with down-regulated CCR activity demonstrated reduced lignin content and abnormal phenotypes, such as collapsed vessels, stunted growth, and abnormal leaf development. See, e.g., Goujon et al., Planta 217:218-228 (2003).
7. Down-Regulation of CAD
CAD catalyzes the final step of monolignal synthesis, i.e., the reduction of cinnamaldehydes to cinnamyl alcohols, and cad mutant plants and genetically-modified plants have been studied. An unusal cad mutant pine possessed an altered lignin composition, even to the extent of incorporating an unusual phenolic monomer. See Ralph et al., Science 277:235-239 (1997). Although the lignin of plants with low CAD activity is more extractable in alkali (see, e.g., Halpin et al., Plant J. 6:339-350 (1994)), lignin content is only slightly effected by the down-regulation of CAD activity (see Pilate et al., Nature Biotechnol. 20:607-612 (2002)).
8. Up- and Down-Regulation of Peroxidases
Peroxidases are believed to catalize the polymerization of lignin, yet no definitive proof have been presented for the involvement of any specific isozyme in vivo. See Baucher et al., supra. This is mainly due to both the large number of genes which encode peroxidases and the low substrate specificity of peroxidases. Although no change in lignin content and composition was observed in transgenic tobacco plants deficient in a major anionic peroxidase (see Lagrimini et al., Plant Physiol. 114:1187-1196 (1997)), a transgenic poplar with reduced anionic peroxidase activity demonstrated some reduced lignin content and altered lignin composition (see Yahong et al., In Molecular Breeding of Woody Plants (Progress in Biotechnology Series, Vol. 18), Elsevier Science, Amsterdam (2001)). Likewise, overexpression of peroxidase genes in poplar produced poor results, with no effect on overall plant phenotype or lignin content. See, e.g., Christensen et al., Plant Mol. Biol. 47:581-593 (2001).
9. Down-Regulation of Laccases
The role of laccases in the lignification process is still very much unclear. It is believed laccases participate in lignin polymerization. See, e.g., McDougall et al., Planta 194:9-14 (1994). However, transgenic Liriodendron (see Dean et al., In Lignin and Lignan Biosynthesis (ACS Symposium Series, Vol. 697), American Chemical Society, Washington, D.C.) and poplar (see Ranocha et al., Plant Physiol. 129:145-155 (2002)) down-regulating laccase had no altered phenotype nor any change in lignin content or composition.
As described above, many different transgenic plants are now available with altered lignin content, altered lignin composition or structure, or both. However, these experiments have few identified transgenic plants with distinct advantages in industrial operations, such as commercial pulping and papermaking. Additionally, these widely-divergent experimental results demonstrate the uncertainty apparent in the attempted modulation of the lignification process in transgenic plants.
C. Expression Profiling and Microarray Analysis of Monolignol Synthesis, Monolignol Transport, and Lignin Polymerization
The multigenic control of the lignification process presents difficulties in determining the genes responsible for phenotypic determination. One major obstacle to identifying genes and gene expression differences that contribute to phenotype in plants is the difficulty with which the expression of more than a handful of genes can be studied concurrently. Another difficulty in identifying and understanding gene expression and the interrelationship of the genes that contribute to plant phenotype is the high degree of sensitivity to environmental factors that plants demonstrate.
There have been recent advances using genome-wide expression profiling. In particular, the use of DNA microarrays has been useful to examine the expression of a large number of genes in a single experiment. Several studies of plant gene responses to developmental and environmental stimuli have been conducted using expression profiling. For example, microarray analysis was employed to study gene expression during fruit ripening in strawberry, Aharoni et al., Plant Physiol. 129:1019-1031 (2002), wound response in Arabodopsis, Cheong et al., Plant Physiol. 129:661-7 (2002), pathogen response in Arabodopsis, Schenk et al., Proc. Nat'l Acad. Sci. 97:11655-60 (2000), and auxin response in soybean, Thibaud-Nissen et al., Plant Physiol. 132:118. Whetten et al., Plant Mol. Biol. 47:275-91 (2001) discloses expression profiling of cell wall biosynthetic genes in Pinus taeda L. using cDNA probes. Whetten et al. examined genes which were differentially expressed between differentiating juvenile and mature secondary xylem. Additionally, to determine the effect of certain environmental stimuli on gene expression, gene expression in compression wood was compared to normal wood. 156 of the 2300 elements examined showed differential expression. Whetten, supra at 285. Comparison of juvenile wood to mature wood showed 188 elements as differentially expressed. Id. at 286.
Although expression profiling and, in particular, DNA microarrays provide a convenient tool for genome-wide expression analysis, their use has been limited to organisms for which the complete genome sequence or a large cDNA collection is available. See Hertzberg et al., Proc. Nat'l Acad. Sci. 98:14732-7 (2001a), Hertzberg et al., Plant J., 25:585 (2001b). For example, Whetten, supra, states, “A more complete analysis of this interesting question awaits the completion of a larger set of both pine and poplar ESTs.” Whetten et al. at 286. Furthermore, microarrays comprising cDNA or EST probes may not be able to distinguish genes of the same family because of sequence similarities among the genes. That is, cDNAs or ESTs, when used as microarray probes, may bind to more than one gene of the same family.
Methods of manipulating gene expression to yield a plant with a more desirable phenotype would be facilitated by a better understanding of monolignol synthesis, monolignol transport, and lignin polymerization gene expression in various types of plant tissue, at different stages of plant development, and upon stimulation by different environmental cues. The ability to control plant architecture and agronomically important traits would be improved by a better understanding of how monolignol synthesis, monolignol transport, and lignin polymerization gene expression effects formation of plant tissues and how plant growth and the lignification process are connected. Among the large number of genes, the expression of which can change during development of a plant, only a fraction are likely to effect phenotypic changes during any given stage of the plant development.